Antisense oligonucleotides for aromatase inhibition

ABSTRACT

The invention relates to an antisense oligonucleotide suitable for inhibiting the expression of aromatase, the antisense oligonucleotide being obtainable by the following steps: 
     (a) construction of antisense oligonucleotides along the entire length of coding and regulatory regions of an aromatase DNA and/or transcripts thereof, the antisense oligonucleotides overlapping; 
     (b) incubation of an aromatase-expressing cell with one or more of the antisense oligonucleotides of (a); and 
     (c) detection of the inhibition of the aromatase expression as usual, as well as identification of the antisense oligonucleotide(s) responsible for this. Furthermore, the invention relates to a process for preparing such an antisense oligonucleotides as well as its use.

I. FIELD OF THE INVENTION

The invention relates to antisense oligonucleotides which are suitablefor inhibiting the expression of aromatase, a process for the productionthereof as well as their use.

II. BACKGROUND OF THE INVENTION

Aromatase belongs to the cytochrome p450 enzyme family. Aromatase is thekey enzyme in the estrogen biosynthesis. It converts the male sexhormones (androgens) into the female ones (estrogens). The latter aregrowth factors for a plurality of tumors, particularly those of ovaries,endometrium and breast.

For treating the above tumors, it is tried to inhibit the estrogenbiosynthesis. Aromatase inhibitors are often used for this purpose.However, they have not shown satisfactory results by now, particularlythey are lacking specificity.

Therefore, it is the object of the present invention to provide apreparation by which aromatase can be inhibited specifically.

According to the invention this is achieved by providing an antisenseoligonucleotide which prevents the expression of aromatase by attachmentto aromatase DNA and/or mRNA. The expression "antisense" is generallyknown and refers to a complementarily of the oligonucleotide sequence tothe region of aromatase DNA and/or mRNA.

III. SUMMARY OF THE INVENTION

The present invention is directed to an antisense oligonucleotidesuitable for inhibiting the expression of aromatase, the antisenseoligonucleotide being obtainable by the following steps:

(a) construction of antisense oligonucleotides along the entire lengthof coding and regulatory regions of an aromatase DNA and/or transcriptsthereof, the antisense oligonucleotides overlapping;

(b) incubation of an aromatase-expressing cell with one or more of theantisense oligonucleotides of (a); and

(c) detection of the inhibition of the aromatase expression as usual, aswell as identification of the antisense oligonucleotide(s) responsiblefor this.

The present invention also directed to a process for preparing such anantisense oligonucleotides as well to methods for its use.

IV. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts a Northern Blot reflecting the effect of an antisenseoligonucleotide according to the invention on the expression ofaromatase mRNA.

FIG. 1B depicts a densiometric quantification of the Northern Blot shownin FIG. 1A.

FIG. 2A depicts the effect of an antisense oligonucleotide according tothe invention on the aromatase activity.

FIG. 2B depicts the effect of an antisense oligonucleotide according tothe invention on the aromatase protein.

V. DESCRIPTION OF THE INVENTION

An antisense oligonucleotide according to the invention can be preparedas usual. A process proves to be favorable which comprises the followingsteps:

(a) construction of antisense oligonucleotides along the entire lengthof coding and regulatory regions of aromatase DNA and/or transcriptsthereof, the antisense oligonucleotides overlapping;

(b) incubation of an aromatase-expressing cell with one or more of theantisense oligonucleotides of (a); and

(c) detection of the inhibition of the aromatase expression as usual, aswell as identification of the antisense oligonucleotide(s) responsiblefor this.

An antisense oligonucleotide according to the invention may havediffering lengths. Lengths of 20 to 30 nucleotides are preferred.Furthermore, the antisense oligonucleotide may have variations in itssugar and phosphate components each. The sugar component conceivable isdeoxyribose, ribose or a chemical variant thereof, for example. Thephosphate component may be, e.g., ortho-phosphoric acid diester or achemical variant thereof. A preferred antisense oligonucleotide containsdeoxyribose as sugar component and ortho-phosphoric acid diester asphosphate component.

In the above step (a), antisense oligonucleotides are constructed alongthe entire length of coding and regulatory regions of an aromatase DNAand/or transcripts thereof. An aromatase DNA and transcripts thereof areknown from Mahendroo et al., 1993, J. Biol. Chem.₁₃ :19463-19470, forexample. Such a DNA comprises 9 coding exons (exons II-X). Furthermore,several exons I (exons I.1.I.4) exist which are non-coding, transcribedin tissue-specific manner and thus have regulatory functions. Thevarious aromatase transcripts (mRNA) are equal in the translated region(exons II-X) but differ in a tissue-specific manner in the 5'untranslated region (exon I).

Antisense oligonucleotides are constructed as usual. It is favorable toconstruct them in overlapping fashion. This facilities the localizationof sequences inhibiting an aromatase expression. Also, such sequencesare to be searched for especially in exon II (5' translated region ofmRNA) and exons I.1-I.4 (5' untranslated region of mRNA) of an aromataseDNA as well as in the 3' untranslated region of an aromatase mRNA.

A preferred antisense oligonucleotide is one having a partial sequenceof exon II of an aromatase DNA, particularly preferably with thefollowing sequence:

    3'-TTCTACCAAAACCTTTACGA-5' (SEQ ID NO:1)

Another preferred antisense oligonucleotide is one having a partialsequence of exon I.1 of an aromatase DNA, particularly preferably withthe following sequence:

    3'-CCTCCCGACTTGTGCACCTC-5' (SEQ ID NO:2).

Another preferred antisense oligonucleotide is one having a partialsequence of exon I.2 of an aromatase DNA, particularly preferably withthe following sequence:

    3-GTAGTCTCTCGGAGGGGAGG-5' (SEQ ID NO:3).

Further preferred antisense oligonucleotides are those having in eachcase one partial sequence of exon I.3 and exon I.4 respectively, of anaromatase DNA. Also, an antisense oligonucleotide which has a partialsequence of the 3' un translated region of an aromatase mRNA ispreferred.

The sequence of an antisense oligonucleotide according to the inventionmay be fully complementary to the sequence, to be bonded, of anaromatase DNA and/or mRNA. On the other hand, the antisenseoligonucleotide may also contain one or more nucleotides which are notcomplementary to the corresponding nucleotides of the sequence to bebonded. In addition, the antisense oligonucleotide may have a sequenceencoding for a functionality such as RNase activity. Ellis and Rogers,1993, Nucleic Acids Res. 21:5171-5178. Such an antisense oligonucleotideshows in an especially advantageous manner when the bonded aromatasemRNA is decomposed. It also represents a subject matter of the presentinvention.

In the above step (b), an aromatase-expressing cell is incubated withone or more of the antisense oligonucleotides according to theinvention. The resulting inhibition of the aromatase expression is thendetected, and the antisense oligonucleotide(s) responsible for theinhibition are identified (above step (c)).

The expression "aromatase-expressing cell" comprises any cells and cellagglomerations capable of expressing aromatase. They include, e.g.,cells of human placenta, furthermore cells of gonads and adipose tissueas well as the corresponding organs and tissues as such. Cells of thechoriocarcinoma cell line JEG-3 are preferred. This cell line isobtainable from the American Type Culture Collection under HTB 36.

An aromatase expression is detected by common methods. They include,e.g., determinations of the aromatase activity, testosterone orandrostenedione, for example, being used as the substrate and theformation of 17β-estradiol (E2) being determined, see, Example 2, infra,of the aromatase protein via the method of Bradford, 1976, Anal.Biochem. 72:248-254, followed by Western blot, or of the aromatase mRNAin a Northern blot, a DNA sample specific to aromatase mRNA, e.g., cDNA,being used. See, Example 2, infra.

The expression of aromatase is effected in the above cells or cellagglomerations by induction and enhanced in the case of JEG-3 cells,respectively. For this purpose, it is possible to use conventionaladenylate cyclase stimulators, e.g,. human chorio gonadotrophin (hCG),or membrane-permeating analogues of cyclic AMP (cAMP) such as dibutyrylcAMP (dbcAMP). The individual concentrations are determined by a personskilled in the art by means of standard tests or follow from theliterature. Nebert and Gonzales, 1987, Ann. Rev. Biochem. 56:945-993.

The above cells or cell agglomerations are incubated with one or more ofthe antisense oligonucleotides according to the invention as usual. Theindividual concentrations are determined by the person skilled in theart by means of standard tests. Furthermore, the aromataseexpression-inhibiting effect of the antisense oligonucleotide(s) can bedetermined directly. The difference from the aromatase expression ofcells or symplasms incubated with and without antisense oligonucleotidesis employed for this purpose. The aromatase expression is determined asdescribed above.

Antisense oligonucleotides according to the invention are perfectlysuitable for the inhibition of aromatase expression. Thus, theyrepresent preparations for inhibiting the estrogen biosynthesis inwell-calculated fashion. This offers new possibilities of being able totreat by means of gene therapy various diseases linked with the estrogenbiosynthesis, particularly tumor diseases.

Antisense oligonucleotides according to the invention can be given aperson as such, individually or in combination. However, they can alsobe expressed within the person by means of an expression vectorcontaining sequences encoding for them.

The invention is explained by the examples. The following preparationsand examples are given to enable those skilled in the art to moreclearly understand and to practice the present invention. The presentinvention is not limited in scope by the exemplified embodiments, whichare intended as illustrations of single aspects of the invention only,and methods which are functionally equivalent are within the scope ofthe invention. Indeed, various modifications of the invention inaddition to those described herein will become apparent to those skilledin the art from the foregoing description and accompanying drawings.Such modifications are intended to fall within the scope of the appendedclaims.

VI. EXAMPLES A. Example 1 Construction of an Antisense Oligonucleotide

In the region of nucleotides 36-55, an aromatase mRNA comprising the5'-translated region (exon II) includes the following sequence:

    5'-GTCAAGGAACACAAGATGTTTTGGAAATGCTGAACCCGATACA-3' (SEQ ID NO:4).

As far as this sequence is concerned, an antisense oligonucleotide ofthe following sequence was constructed. A generally available synthesisapparatus (Applied Biosystems) was used:

    3'-TTCTACCAAAACCTTTACGA-5' (SEQ ID NO:1).

B. Example 2 Inhibition of Aromatase Expression

Semi-confluent JEG-3 cells, see, supra, were incubated in 2 charges in astandard medium for 14 hours each. The medium of one charge did notcontain dbcAMP. See, supra. The cells are referred to as "untreatedcells C" below. The medium of the other charge contained 1 mM of dbcAMP.These cells are referred to as "stimulated cells S" below.

Then, 100 μ/ml of the antisense oligonucleotide of Example 1 were addedin each case three times to the untreated cells (C) and the stimulatedcells (S). The additions were made 14, 16 and 18 hours after the timewhen dbcAMP had been added to the stimulated cells (S). 24 hours afterthe above time all cells were harvested separately and analyzed withrespect to the following aspects:

(I) Aromatase mRNA

(II) Aromatase activity; Aromatase protein

As to (I) Aromatase mRNA:

Entire RNA was isolated each from the harvested cells as usual. This RNAwas analyzed twice (FIG. 1A and 1B):

(A) Northern Blot (FIG. 1A):

The RNA (12 μg) was subjected to a conventional Northern blot in whichit was hybridized against an aromatase cDNA fragment which comprisesexon II partially, exons III and IV completely as well as exon Vpartially (FIG. 1A, panel a). Furthermore, the RNA was hybridizedagainst a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA fragmentas control (FIG. 1A, panel b). The indication C refers to untreatedcells without antisense oligonucleotide, ^(C) AS denotes untreated cellswith antisense oligonucleotide, S represents stimulated cells withoutantisense oligonucleotide and ^(S) AS stands for stimulated cells withantisense oligonucleotide.

(B) Densitometric quantification (FIG. 1B):

The autoradiography signals of aromatase mRNA in FIG. 1A were set to100% in those cases in which no antisense oligonucleotide treatment (C,S open columns) was given and compared with the signals obtained in thecase of cells with antisense oligonucleotide treatment (^(C) AS, ^(S)AS; dashed columns).

Thus, FIG. 1A discloses that after the addition of the antisenseoligonucleotide, the amount of aromatase mRNA was reduced by about 60%.Furthermore, an increased degradation of aromatase mRNA was affected.

As to (II) Aromatase activity; Aromatase protein:

(A) Aromatase activity (FIG. 2A):

The harvested cells were taken up in a homogenization buffer, pH 7.4 (10mM potassium phosphate, 150 mM KCL and 10 mM EDTA). The cells wereopened up by ultrasonic treatment and microsomes were obtained aftercentrifugation of the homogenate at 10,000 g for 20 minutes andcentrifugation of the resulting supernatant at 300,000 g for 20 minutes.They were resuspended in a reaction buffer, pH 7.4 (50 mM potassiumphosphate, 2.5 mM glucose-6-phosphate, 0.25 U/ml glucose-6-phosphatedehydrogenase and 10 μM testosterone as substrate). The mixture wasincubated at 37° C. for 5 minutes, and the reaction was started byadding NADPH up to a final concentration of 100 μM. After 5 hours, thereaction was stopped by 5-minute heating to 95° C. and the formation of17β estradiol was determined by a competitive E2 enzyme immunoassay kit(Dianova). In FIG. 2A, the indication S refers to stimulated cellswithout antisense oligonucleotide and ^(S) AS are stimulated cells withantisense oligonucleotide. The former were set to be 100%.

(B) Aromatase protein (FIG. 2B):

70 μg protein each were inserted from the microsomes in an SDS page.Then, the protein was transferred to a PVDF membrane, and the aromataseprotein was identified by a generally available, monospecific polyclonalaromatase antibody.

Thus, FIGS. 2A and 2B discloses that after the addition of the antisenseoligonucleotide the aromatase activity was reduced by about 60%. Inaddition, non-identification of the aromatase protein was affected in aWestern blot.

All references cited within the body of the instant specification arehereby incorporated by reference in their entirety.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 4                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 # 20               TACGA                                                      - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 # 20               ACCTC                                                      - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 # 20               GGAGG                                                      - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 43 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 # 43               ATGTT TTGGAAATGC TGAACCCGAT ACA                            __________________________________________________________________________

What is claimed:
 1. An antisense oligonucleotide, said antisenseoligonucleotide consisting of the sequence:

    3'-TTCTACCAAAACCTTTACGA-5' (SEQ ID NO:1).


2. An antisense oligonucleotide, said antisense oligonucleotideconsisting of the sequence:

    3'-CCTCCCGACTTGTGCACCTC-5' (SEQ ID NO:2).